An innovative approach promises to revolutionize the study of lipid damage during oxidative stress.

Anyone who pays attention to current health news has heard about the importance of antioxidants. We have been told that antioxidants will help prevent or slow the development of a range of conditions, including cancer, heart disease, neurodegenerative diseases, and aging, and there are plenty of companies ready to sell us supplements so that we can achieve these laudable goals. The importance of antioxidants in biology stems from the critical role of oxygen in metabolism. Oxygen is absolutely required for chemical processes that produce energy, but it can also be converted into highly reactive free radicals, collectively known as reactive oxygen species (ROS), that can damage other cellular constituents such as DNA, protein, and lipids. All cells contain an armamentarium of protective antioxidants that react with, and neutralize ROS. However, in certain disease states, these intrinsic defenses may be overwhelmed. The result is a condition known as oxidative stress, which is associated with potentially devastating damage to the cell.

Despite the current antioxidant hype, there are few well controlled clinical studies that support the value of taking supplements to prevent conditions associated with oxidative stress, and some studies suggest that high dose supplements may even be harmful. Thus, understanding the mechanisms of oxidative cell damage is key to developing a rational approach to combating this condition. A prime target of ROS is the polyunsaturated fatty acids (PUFAs) that are a major component of cell membrane phospholipids (Figure 1).

Figure 1. Lipid bilayer comprised primarily of phospholipids. The fatty acid chains of the phospholipids, shown in gray, comprise the center of the bilayer. (Image courtesy of Wikimedia Commons under the GNU Free Documentation License.)

PUFAs are characterized by a long chain of carbon atoms, and the presence of two or more carbon-to-carbon double bonds. The double bonds render PUFAs susceptible to ROS attack, which breaks the carbon chains into fragments that are themselves highly reactive and capable of inflicting further damage. Some of these fragments stay within the cell membrane where they damage other lipids or membrane proteins. Other fragments are free to move throughout the cell, including into the nucleus where they can damage DNA (Figure 2).

Figure 2. ROS damage to membrane phospholipids. A typical phospholipid containing one 16-carbon (black) saturated fatty acid (SFA) and one 18-carbon PUFA with two double bonds (blue). The PUFA has been labeled with a triple bond (red) at the end. ROS attack at the double bond of the PUFA, cleaves the carbon chain and creates reactive groups (orange) which may remain attached to the phospholipid or be free to damage other cellular constituents (lipids, protein, or DNA).

Cell membranes contain a diverse array of different PUFAs (different carbon chain length and number of double bonds), and they can fragment in multiple ways when attacked by ROS. Thus, identifying the exact identities and sites of damage caused by the fragments has been very difficult. Now work from the labs of VICB members Alex Brown, Ned Porter, and Larry Marnett, along with Chuck Lukehart of the Vanderbilt Chemistry Department promises to revolutionize the way lipids and their metabolites are tracked within a cell. This work, supported initially by a VICB Pilot Project grant, and now by an NIH Program Project Grant (National Institute of Environmental Health Sciences), will soon be published in Nature Chemical Biology [S.B. Milne, K.A. Tallman, et al.

Key to the success of the new approach is to label a fatty acid by the introduction of a carbon-to-carbon triple bond at the end of its carbon chain (Figure 2). The labeled fatty acid can then be fed to cells directly or as part of a complete pre-formed phospholipid molecule. The structure of the triple bond is so similar to that of the carbon-to-carbon single bond found normally at the end of a fatty acid chain, that cells readily take up the labeled lipids, incorporate them into membranes, and metabolize them like their native counterparts. Once incorporated, the triple bond provides a “handle” that allows the investigators to retrieve the labeled lipid from the complex cellular environment, and to analyze it for any chemical changes. This is done by the ability of the triple bond to form a complex with cobalt, and by the ability of that complex to bind selectively to a purification column made from modified silica. Other cellular components are washed away, and then a mild chemical reaction breaks the cobalt complex, releasing the labeled lipid unaltered, along with any bound cellular constituents. The fact that triple bonds are not found naturally in cellular molecules guarantees that only the labeled lipid will be retrieved. Furthermore, since the overwhelming majority of biochemical reactions (including oxidations) do not alter triple bonds, retrieval can be made regardless of what other chemical changes have befallen the original labeled molecule. Thus, even if a labeled PUFA chain is fragmented through reaction with ROS, and the fragment containing the label travels to the nucleus and reacts with DNA, this method provides a way to find that molecule and precisely determine its fate.

The Vanderbilt team is now actively investigating the fate of lipids during conditions of oxidative stress, and using this new information to design more effective antioxidants. Their goal is to rationally design a new approach to prevent the damage caused by ROS. In the meantime, it is wise to eat plenty of fresh fruits and vegetables that contain a wealth of natural and safe antioxidants.

Figure 3. The reactive phospholipid fragment shown in Figure 2 has bound to and damaged a cellular molecule (protein or DNA, green). Addition of cobalt (purple) forms a complex with the triple bond at the end of the fragment (red). The complex binds to modified silica, allowing all other cellular components to be washed away. Then the damaged molecule and lipid fragment are recovered for analysis by a mild reaction that breaks the cobalt complex.